Studies on the Respiratory Chain-linked Reduced Nicotinamide Adenine Dinucleotide Dehydrogenase XIII. BINDING SITES OF ROTENONE, PIERICIDIN A, AND AMYTAL IN THE RESPIRATORY CHAIN

摘要: The binding sites of rotenone, piericidin A, and Amytal in the reduced nicotinamide adenine dinucleotide oxidase chain heart have been studied with aid rotenone-6a-14C. Binding rotenone continues a linear manner beyond point maximal inhibition respiration, indicating that is tightly bound not only at specific site NADH dehydrogenase segment respiratory but also other submitochondrial particles. Unspecific by treatment particles presence bovine serum albumin (BSA) further minimized successive washing BSA, minimal removal from site. Rotenone-14C removed denaturation or proteolysis largely repeated extractions anhydrous acetone, which results recovery primarily small amounts less inhibitory oxidation product. Substantial reversal respiration occurs on inhibited BSA. Thus, contrary to expectations, under usual assay conditions specific, stoichiometric, irreversible inhibitor. Studies involving preincubation electron transport particle various unlabeled inhibitors, widely varying chemical type potency, prior addition rotenone-14C for determination its subsequent binding, provide strong evidence it possible distinguish between portion specifically unspecifically bound. Piericidin Amytal, number rotenoids appear react same as because they compete site, competition proportional power compounds tested their concentration nature. Although molar ratio content approximates unity, itself, since extraction enzyme phosphorylating non-phosphorylating labeled nearly complete separation enzyme. Qualitative quantitative differences are evident soluble NADH-coenzyme Q reductase compared reductase, extracted acid-ethanol rotenone-14C, fully active contains little no may be again rotenone.